Br. J. Pharmacol.

(1993), 108, 914-919

"f"

Macmillan Press Ltd, 1993

Degradation of acetylcholine in human airways: role of

butyrylcholinesterase
'X. Norel, *M. Angrisani, C. Labat, I. Gorenne, E. Dulmet, *F. Rossi & C. Brink
CNRS URA 1159, Centre Chirurgical Marie-Lannelongue, 133 av. de la R6sistance, 92350 Le Plessis-Robinson, France and
*Department of Pharmacology and Toxicology, First Faculty of Medicine and Surgery, via Costantinopoli, 16, 80138 Naples,
Italy
1 Neostigmine and BW284C51 induced concentration-dependent contractions in human isolated bronchial preparations whereas tetraisopropylpyrophosphoramide (iso-OMPA) was inactive on airway resting tone.
2 Neostigmine (0.1 pM) or iso-OMPA (100 pM) increased acetylcholine sensitivity in human isolated
bronchial preparations but did not alter methacholine or carbachol concentration-effect curves.
3 In the presence of iso-OMPA (10 pM) the bronchial rings were more sensitive to neostigmine. The
pD2 values were, control: 6.05 0.15 and treated: 6.91 0.14.
4 Neostigmine or iso-OMPA retarded the degradation of acetylcholine when this substrate was
exogenously added to human isolated airways. A marked reduction of acetylcholine degradation was
observed in the presence of both inhibitors. Exogenous butyrylcholine degradation was prevented by
iso-OMPA (1O pM) but not by neostigmine (O.1 pM).
5
These results suggest the presence of butyrylcholinesterase activity in human bronchial muscle and
this enzyme may co-regulate the degradation of acetylcholine in this tissue.
Keywords: Human bronchus; butyrylcholinesterase; acetylcholinesterase; acetylcholine; butyrylcholine; tetraisopropylpyrophosphoramide; neostigmine

Introduction
The inherent tone observed in human airways is maintained
by the local actions of neuronal inputs derived from the
parasympathetic nervous system. This system regulates airway calibre by activation of the post-synaptic muscarinic
sites on airway smooth muscle via the release of the neurotransmitter, acetylcholine (ACh). Enzymatic degradation of
ACh occurs through the action of cholinesterases. Two
different cholinesterases have been described in many tissues
(Mann, 1971; Aas et al., 1986; Chatonnet & Lockridge, 1989;
Small et al., 1990) namely, acetylcholinesterase (AChE: EC
3.1.1.7) and butyrylcholinesterase (BChE: EC 3.1.1.8). Previous investigators (Mittag et al., 1971; Small et al., 1990;
Adler et al., 1991) have shown that BChE represents a
considerable fraction of the cholinesterase pool in smooth
muscle. While the function of AChE at the cholinergic
neuronal synapse in airway muscles is to regulate the duration of action of ACh (Adler & Filbert, 1990), the physiological role of BChE has not been systematically examined in
human airways. In vivo, treatment with cholinesterase inhibitors leads to an increase in resistance to airflow in animals
(Pauluhn et al., 1987) and in man (Haber et al., 1987).
Recently, Adler and co-workers (1991) proposed that BChE
may participate in the co-regulation of the duration of action
of ACh in dog airways in vitro. These investigators demonstrated that the contractions induced by electrical field stimulation were enhanced and prolonged in tissues treated with
the specific BChE inhibitor, tetraisopropylpyrophosphoramide (iso-OMPA; Atack et al., 1989). While Ito and coworkers (1989) have demonstrated that physostigmine, a
non-specific cholinesterase inhibitor, enhanced the response
to electrical field stimulation in human isolated bronchial
muscles, few studies have been performed to examine the
relative contribution of these two enzymes on the functional
response of human airways.
The aim of this study was to determine the sensitivity of
human bronchial preparations to exogenous and endogenous
ACh in the presence of specific cholinesterase inhibitors. A
Author for correspondence.

measurement of the degradation of exogenous ACh or butyrylcholine (BuCh: specific substrate for BChE) were also performed in order to compare the activity of AChE and BChE
in human isolated bronchial preparations. These studies were
undertaken to test the hypothesis that BChE may be a
co-regulator of ACh activity in human airways.

Methods

Isolated preparations
Human lung tissue was obtained from 34 male and 6 female
patients who had undergone surgery for lung carcinoma. The
mean age was 56.20 1.15 years. After the resection of a
lung or a lobe, parts of the bronchus were dissected free from
parenchymal lung tissue and placed in Tyrode solution at
4C for 12 h. All experiments were performed on 161 subsegmental bronchial preparations derived from 40 lung samples
(n); these bronchial ring preparations were of 2-4 mm internal diameter and weighed 63.09 2.84 mg. Values are means
s.e.mean.

Physiological studies (contractions)

Bronchial ring preparations were set up in the 10 ml organ
baths containing Tyrode solution (concentration mM): NaCl
139.2, KCl 2.7, CaCl2 1.8, MgC12 0.49, NaHCO3 11.9, NaH2
P04, 0.4 and glucose 5.5; pH 7.4; gassed with 95% 02/5%
CO2 and maintained at 37C. The preparations were placed
under initial loads (2-3 g) depending on their size.
These loads ensured that responses to contractile agonists
were maximal. Isometric force displacement transducers
(Narco F-60) and physiographs (Linseis) were used to record
the changes in force. The tissues were allowed to equilibrate
for 90 min and the bath fluid was exchanged every 15 min
with fresh Tyrode solution.
After this period, all bronchial preparations were contracted with ACh (100 gM). The tissues were washed with

HUMAN AIRWAYS AND BUTYRYLCHOLINESTERASE

fresh Tyrode solution and allowed to return passively to their

resting tone. When resting tone was established, two different
protocols were followed. In the first protocol, each cholinesterase inhibitor (neostigmine, BW284C51 or iso-OMPA)
was added to the bath fluid at 10 min intervals in a cumulative fashion, beginning with the lowest concentration. In the
second protocol, the preparations were incubated for 30 min
in Tyrode solution containing either neostigmine or isoOMPA, and subsequently a cholinoceptor agonist-response
relationship was determined. Cumulative concentration-effect
curves to neostigmine were also produced in the tissues after
a 30 min incubation with iso-OMPA. Atropine 1 fM was
added 30 min before or at the end of the cholinesterase
inhibitors concentration-effect curves.

Biochemical studies (enzyme activities)

Bronchial rings were placed in 10 ml tubes containing 5 ml
Tyrode solution gassed with 95% 02/5% CO2 and maintained at 37C. After a 60 min equilibration period, the
preparations were pre-incubated for 30 min in Tyrode solution or Tyrode solution containing cholinesterase inhibitors
(neostigmine or iso-OMPA) at different concentrations. ACh
or BuCh (0.5 ;tmol) was then added to each tube: 1 h and 3 h
after this substrate challenge, a 30 .d sample of supernatant
was collected and stored at - 20'C. The cholinesterase inhibitors were always present in appropriate tubes during the 3 h
following the pre-incubation period. Measurements of choline, ACh and BuCh in the supernatants were performed
according to the technique of Israel & Lesbats (1985). This
assay involved hydrolysis of ACh by AChE to give choline,
which was oxidized to betaine by choline oxidase with H202
production. Using this technique for measurement of BuCh,
the AChE was markedly less efficient in degrading this substrate. In the presence of luminol, H202 and horseradish
peroxidase (HRP), light emission was generated and recorded
by a photomultiplier (1250 LKB-Wallac) connected to a
potentiometric recorder (2210-032 LKB-Wallac). The assay
was carried out in a microcuvette containing 250 fl of phosphate buffer (Na2HPO4/NaH2PO4 0.2 M; pH 8.6) and 10 fd of
a mixture containing choline oxidase (62.5 u ml-'), horseradish peroxidase (0.5 g 1-') and luminol (0.5 mM). The baseline level of luminescence was obtained using these enzymes
and buffer. When the samples or choline standards (10 yt)
were added to this mixture a peak of luminescence was
induced. After the baseline had been re-established, 10 l of
purified AChE (less than 250 u ml ') was added to determine
the ACh or BuCh present in samples or standards. Standards
were made up daily, and were tested for each lung sample;
the relationship between peak luminometer deflection and
amount of added standard in the assay system was linear
over the range (4 pmol-2 nmol). Sample values were derived
from standard curves. In each lung sample experiment, stock
solutions of ACh or BuCh were prepared twice: initially for
the tissue incubations in the tubes and again for standards in
the chemiluminescence technique.

Data analysis
Physiological studies (contractions) The changes in force
were measured from isometric recordings and expressed in
grams (g) or as a percentage of the initial contraction
induced by ACh (100JLM). The maximal response (E,,) was
induced with a cholinoceptor agonist, neostigmine or BW284
C51 and the EC50 values were interpolated from the individual concentration-effect curves. The EC50 values were transformed into pD2 values, that is, negative logarithms of EC50
values.
Biochemical studies (enzyme activities) The amounts of
ACh or BuCh remaining in the supernatants after 1 or 3 h as
well as the quantities obtained after treatment of tissues with
enzyme inhibitors were expressed in pmol. The protection of

915

ACh or BuCh degradation by the different inhibitors was

calculated from the following formula: 100 x (Sc - Sb)/(Sa Sb) where S,, = 0.5 gmol of ACh or BuCh, Sb = the quantity
(pmol) of ACh or BuCh remaining after 3 h of incubation
with Tyrode solution, Sc = ptmol of ACh or BuCh remaining
after 3 h of incubation with a cholinesterase inhibitor.
All results are expressed as means s.e.mean. Statistical
analysis was performed using Multirange ANOVA followed
by a post-hoc test (Least Significant Difference) with a confidence level of 95% and taking into account the preparations derived from the same or different lung samples.

Drugs
The drugs and their sources were: acetylcholine chloride,
butyrylcholine chloride, choline chloride (three times crystallized), methacholine chloride (acetyl-p-methylcholine chloride), carbachol (carbamylcholine chloride), atropine sulphate,
iso-OMPA (tetraisopropylpyrophosphoramide), BW284C51 (1,
5-bis (4-allyldimethylammoniumphenyl) pentane-3-one dibromide), Trizma buffer solution [Tris(hydroxymethyl) aminomethane], choline oxidase from Alcaligenes species, peroxidase
HRP II (Horseradish peroxidase type II) (Sigma Chemical
Co., St. Louis, MO, U.S.A.). Neostigmine methylsulphate
(Prostigmine; Roche, 92521 Neuilly sur Seine, France).
Luminol (5-amino-l ,2,3,4 tetrahydrophtalazindion- 1,4) (Merck,
Schuchardt 8011 Hohenbrunn bei Miinchen, Germany). Acetylcholinesterase from Electrophorus electricus was obtained
from Boehringer Mannheim GmbH (Germany) and purified
by passing through a Sephadex G50 coarse column (Pharmacia, Uppsala, Sweden).
All cholinoceptor agonists and BW284C51 were dissolved in
Tyrode solution and enzymes were dissolved in distilled water.
Iso-OMPA (0.1 M) was dissolved in 100% ethanol. Subsequent
dilutions of each drug and neostigmine were made in Tyrode
solution. A stock solution of luminol (1 mM) was prepared by
dissolving 18 mg in a few drops of 1 M NaOH, and the volume
was adjusted to 100 ml with 0.2 M Tris buffer (final pH of 8.6).

Results

Physiological studies (contractions)

The data presented in Figure 1 demonstrate that neostigmine
produced concentration-dependent contractions in human isolated bronchial preparations whereas iso-OMPA did not provoke contractions under similar experimental conditions. The
pD2 values for neostigmine and BW284C51 on human bronchial preparations were: 6.46 0.23 and 6.73 0.22, respectively,
while the maximal effect of neostigmine was two fold greater
than that of BW284C51. These data were obtained in paired
preparations derived from 4 lung samples. The neostigmine
and BW284C51 responses were completely reversed or inhibited by atropine (1 pM). Pretreatment of human bronchial
muscles with neostigmine (0.1 M; 30 min; Figure 2a and Table
1) or iso-OMPA (100IM; 30 min; Figure 2b and Table 1)
significantly shifted to the left the ACh curves in these
preparations without modifying the reactivity (Em,,). In contrast, these inhibitors at the same concentrations failed to alter
the methacholine or carbachol concentration-effect curves
(Table 1). Iso-OMPA (10 EM) did not shift ACh curves (Table
1).
The concentration-effect curves obtained with neostigmine
were shifted to the left when the bronchial preparations were
pretreated with iso-OMPA (30 min; 10 pm; Figure 3 and Table
1).

Biochemical studies (enzyme activities)

Three hours after the addition of exogenous ACh (0.5 Amol)
or BuCh (0.5 pmol) to human isolated bronchial ring preparations, a marked reduction in the quantities of these substrates

X. NOREL et al.

916

150 r-

250 r

200

CL
0o
I-

150 _-

0-~~

10O-

0
C)

I/

50

50 -

u
en
0
c
0
CL
cn

00

--ll~~~~~~~~~~~~~~~~~~~~~~~~~~~

(0
4)

%-0

Cholinesterase inhibitor [-log Ml

Figure 1 Effect of cholinesterase inhibitors on basal tone in human
isolated bronchial preparations. Cumulative concentration-effect
curves were constructed with neostigmine (0; n = 4), BW284C51
(0, n = 4) or tetraisopropylpyrophosphoramide (iso-OMPA, 0;
n = 5). Each response was expressed as a percentage of the acetylcholine (ACh, 100 tM) contraction induced prior to the cumulativecurve. Values are means with s.e.mean shown by vertical bars.

-C
0
c

7I

4
3

250r

.)

0
Cc

0o

200 p

I---I----

150 -

100

was observed: ACh (0.10 0.04 pmol; n = 7) and BuCh

(0.05 0.03 ;mol; n = 4). This reduction was also detectable at
1 h: ACh (0.25 0.07 pmol; n = 7) and BuCh (0.17 0.06

mrol; n = 4).
In paired preparations which had been pretreated for 30 min
with neostigmine (0.1 tIM), prior to the addition of ACh, or in
tissues treated with iso-OMPA (10 tM), before the BuCh
challenge, a small or no reduction in the quantities of these
substrates was detected. Thus at these concentrations, the
enzyme inhibitors prevented the hydrolysis of the appropriate
substrate (Table 2). In experiments with BuCh, this substrate
was degraded even if tissues were treated with neostigmine
(Figure 4 and Table 2). The protection of ACh or BuCh by
each inhibitor at the concentrations used and neostigmine
10 gLM are shown in Table 2. The ACh remaining at 1 h after
treatment with neostigmine (0.1 LM) or iso-OMPA (100 gM)
were: 0.49 0.05 grmol and 0.50 0.06 grmol, respectively.
However, in tissues treated with both neostigmine (0.1 M) and
iso-OMPA (100 AM), the quantity of ACh detected was 0.59
0.05 pmol (n = 7 paired lung samples). This quantity was significantly greater than those detected when tissues were treated
with the individual inhibitors. The ACh results obtained after
3 h with the inhibitors were: 0.32 0.06 pmol and 0.43 0.06
jtmol for neostigmine (0.1 M) and iso-OMPA (100 PM),
respectively. The ACh remaining after 3 h in tissues treated
with a combination of both inhibitors at these same concentrations was: 0.51 0.05 grmol. These latter data were greater
than results obtained in supernatants in the presence of one
inhibitor. When lower concentrations of these enzyme inhibitors were used under the same experimental conditions, a more
pronounced alteration was observed, that is, both inhibitors in

50

--ll

Acetylcholine [-log Ml
Figure 2 Acetylcholine (ACh) concentration-effect curves produced
in human isolated bronchial preparations after 30 min incubation
with Tyrode solution (@) or cholinesterase inhibitors (0). (a) Neostigmine (0.1 IJM; n = 6) (b) tetraisopropylpyrophosphoramide (isoOMPA, 100 gIM; n = 4). Each response was expressed as a percentage
of the ACh (100tiM) contraction induced prior to the incubation.
Values are means s.e.mean from paired lung samples.

combination significantly protected ACh against degradation

at 1 h (Figure 5a) and at 3 h (Figure 5b).
Choline measurements for each of the preceding experiments
were exactly the reverse of all measurements of ACh or BuCh.
The choline content increased in supernatants when degradation of ACh or BuCh occurred specifically when tissues were
not exposed to cholinesterase inhibitors (data not shown).

HUMAN AIRWAYS AND BUTYRYLCHOLINESTERASE

917

Table 1 Effects of cholinesterase inhibitors on acetylcholine, carbachol, methacholine and neostigmine concentration-effect curves in
human isolated bronchial preparations
ACh (100 gM)
response (g)

Treatment

Tyrode
Iso-OMPA (10 gM)
Iso-OMPA (100 SM)
Neostigmine (0.1 ItM)

17
5
6
6

2.30 0.56

Tyrode
Iso-OMPA (100IM)

4
4

1.86 0.50
1.90 0.38

Tyrode
Neostigmine (0.1 gtM)

3
3

0.95 0.40

Tyrode
Iso-OMPA (10 gM)

5
5

1.770.27

Agonist
E,, (g)

pD2value

Acetylcholine
4.70 0.36
2.42 0.36
4.91 0.12
2.30 0.55
5.57 0.26*
3.14 0.69
5.68 0.20*
2.90 0.84
Carbachol
6.34 0.03
2.88 0.74
6.50 0.12
2.65 0.55
Methacholine
6.76 0.03
1.22 0.43
6.60 0.26
1.63 0.49
Neostigmine
1.42 0.27
6.05 0.15
2.41 0.18
6.91 0.14*

1.74 0.29

1.420.28
1.780.48

1.470.53
1.46 0.38

Acetylcholine (ACh, 100 JtM) response was obtained prior to treatment. Tissues were exposed to Tyrode solution or to Tyrode solution
containing cholinesterase inhibitors (30 min). E.,8 indicates the maximal contraction produced by the agonist expressed in grams (g).
Values are means s.e.mean, n indicates the number of lung samples.
*Values significantly different when results from control and treated tissues were compared (P <0.05, ANOVA).

Table 2 Protection against the degradation of acetylcholine

(ACh) and butyrylcholine (BuCh) in human isolated
bronchial preparations

200 k-

Neostigmine
0.1
0.01
150

10

Iso-OMPA (AM)
100
10

Substrate
ACh
2116

Cl)
W

6617 102 13 19 05 96 22
(4)
(7)
(4)
(4)
(7)
3 10 2504 5206 94 09 106 10
(3)
(4)
(4)
(3)
(7)

0.Cl)

BuCh

Results are expressed as percent protection against the

degradation of ACh or BuCh. Protection was evaluated 3 h
after the addition of 0.5 pmol of substrate. Values are means
with s.e.mean. Number of lung samples used is indicated in
parentheses.

oa
0
,

(pM)

h.2
0
._

C-)0

5C 7

0.6f-

0.5F

,L

rV-

Neostigmine [-log Ml
Figure 3 Effect of tetraisopropylpyrophosphoramide (iso-OMPA)
on neostigmine concentration-effect curves in human isolated bronchial preparations. Data are derived from curves produced after a
30min incubation with Tyrode solution (m) or Tyrode solution
containing iso-OMPA (10 pM; 0). Each response was expressed as a
percentage of the acetylcholine (ACh, 100tpM) contraction induced

prior to the incubation. Values are means s.e.mean from paired

lung samples. Preparations were derived from 5 lungs.

E
0
C

0.4F
0.3-

IC.

0.2
0.1

0.0'

Discussion
The results presented in this paper demonstrate that two functional cholinesterase enzyme pools are present in human
bronchial muscle preparations. Hydrolysis of ACh is achieved
by the action of endogenous AChE and is inhibited by neostigmine or BW284C51 (Atack et al., 1989; this paper). In addition, BuCh degradation occurs via BChE (Mittag et al., 1971),
an activity which is prevented by pretreatment of the tissues

ri0.01
Cont

0.1

Neo (lM)

10
100
ISO (ILM)

Figure 4 Butyrylcholine (BuCh) detected in supernatants of human

isolated bronchial preparations after 3 h incubation with Tyrode
solution (Cont) or Tyrode solution containing neostigmine (Neo) or
tetraisopropylpyrophosphoramide (Iso). The quantity of BuCh initially added (time zero) was 0.5 pmol. Values are means s.e.mean
of tissues derived from 3-4 lung samples. Values significantly different from (*) control data or (a) neostigmine (0.1 gM) results
(P<0.05, ANOVA).

918

X. NOREL et al.

0.6F
0

0.5F

E 0.41
._

0.3-

u.

0.2 -

or

0.1

Cont

Neo

Iso Neo +

Cont

Neo

Iso Neo +

ISO

Figure 5 Acetylcholine (ACh) detected in supernatants of human

isolated bronchial preparations after 1 h (a) or 3 h incubation (b) in
Tyrode solution (Cont) or Tyrode solution containing: neostigmine
(Neo; 0.01 AM); tetraisopropylpyrophosphoramide (Iso; 10 fAM) or a
combination of both inhibitors at these concentrations (Neo + Iso).
The quantity of ACh initially added (time zero) was 0.5 pmol. Values
shown are means s.e.mean from paired lung samples. Preparations
were derived from 4 lungs. (*) Values significantly different when
compared with the three other values (P< 0.05, ANOVA).

with iso-OMPA (Austin & Berry, 1953; Thomsen et al., 1991;

this paper). In bronchial preparations, the responses induced
with BW284C51 were approximately one half of those responses observed with neostigmine (Figure 1). These results
support the anti-muscarinic effect of BW284C51 as described
by Ambache & Lessin (1955) and Adler et al. (1991). Therefore, we used neostigmine as the AChE inhibitor in all other
protocols. The data obtained in human airways (this paper)
are similar to those results derived from canine tracheal
smooth muscle preparations (Adler & Filbert, 1990; Adler et
al., 1991). These functional studies support the histochemical
findings in which both cholinesterases have been detected in
respiratory smooth muscles from a variety of animal species
(Mann, 1971; Small et al., 1990) including man (Partanen et
al., 1982).
Initial studies dealing with the effects of AChE activity in
airway preparations have shown that inhibition of this enzyme
by agents such as physostigmine and neostigmine increases the
contractile response to exogenous ACh in airway preparations
(Douglas, 1951; De Candole et al., 1953; Daly, 1957; Carlyle,
1963) or to electrical field stimulation (Kirkpatrick & Rooney,
1982; Ito et al., 1989). However, none of these reports distinguished between the relative contributions of the different
cholinesterases involved in the contractions to cholinoceptor
agonists in airway muscles. Recently, Adler and co-workers
(1991) demonstrated that both AChE and BChE play a role in
the co-regulation of canine airways response to electrical field
stimulation. These observations are supported by the results
obtained in human airways (this paper), where the role of
BChE in the ACh contraction can be observed in tissues which
were challenged with increasing concentrations of neostigmine
in the presence of iso-OMPA (see Figure 3). These functional
data (contractions) are supported by the biochemical measurements in bronchial tissues treated with exogenous ACh in the
presence of iso-OMPA (see Figure 5).
While the activities of both enzymes were detected in canine
and human isolated airways, few reports have described the
distribution of these enzymes in respiratory tissues. The suggestion that these enzymes were distributed differently in
different tissues, was based on an early report (Koelle &
Koelle, 1959) which showed that AChE in autonomic ganglia

of the cat was confined to the neural elements, whereas BChE

was restricted to the glial cells. Appleyard & Smith (1989)
showed that BChE was present in ileal smooth muscle,
whereas AChE was detected in nerves of Auerbach's plexus.
Recent evidence derived from the guinea-pig trachea (Small et
al., 1990) indicated that BChE was mainly detected in smooth
muscle cells while AChE was found in nerve fibres running
within the smooth muscle. These data suggest a tissue distribution for the cholinesterases with AChE exhibiting a close
association with neuronal elements, while BChE has an extraneuronal location. Our physiological results (Figure 1) are in
agreement with these reports. Muscarinic M3-receptors are
uniformly distributed on smooth muscle cells of human distal
bronchi (Mak & Barnes, 1990). A dense nerve supply has been
reported throughout the smooth muscle in human airways
(Partanen et al., 1982; Daniel et al., 1986). These reports and
our physiological results suggest that cholinergic nerve endings
are co-localized with AChE, but not with BChE. Excess ACh
(present in tissues treated with neostigmine) may lead to a
diffusion of this neurotransmitter from the parasympathetic
varicosities to the smooth muscle regions where hydrolysis
may occur via BChE. This may explain the increased sensitivity to neostigmine which was observed in human airways
in the presence of iso-OMPA. These data suggest that neurotransmitter hydrolysis may be controlled by a dual enzymatic
process: the primary regulatory, namely AChE, responsible for
the local neuronal synaptic regulation of ACh and BChE a
secondary system, to protect against an increased release of
ACh or against extra-neuronal appearance of ACh.
The results derived from human bronchial preparations,
confirm previously published data in animal tissues concerning
the different substrates for cholinesterases (Mittag et al., 1971;
Adler & Filbert, 1990). ACh is degraded by both AChE and
BChE while BuCh is principally hydrolysed by BChE (Table
2). In some protocols the supernatant samples obtained following cholinesterase inhibitor treatments contained slightly
higher quantities of the exogenously added substrates (0.5
imol ACh or BuCh). This may have been due to protection of
the endogenous production of ACh in presence of cholinesterases inhibitors.
Kirkpatrick & Rooney (1982) have suggested that in bovine
tracheal muscle, neostigmine may act not only by preventing
endogenous ACh degradation but also by stimulating release
of endogenous ACh from nerve terminals. This notion was
supported by the potentiation of maximal contractions to
histamine, carbachol and acetylcholine in the bovine tracheal
muscle. However, the sensitivity to these agents were unaffected. In contrast, the results presented here demonstrate
that a higher concentration of neostigmine shifted the ACh
concentration-effect curves to the left, while the methacholine
curves were unaltered.
Iso-OMPA (10 jiM) was a selective inhibitor of BChE. However, a higher concentration of iso-OMPA (100ftM) appeared
not to be selective, since subsequent to 3 h of incubation, the
protection against ACh enzymatic degradation was maximal
(see Table 2). However, treatment of tissues for a shorter
period (30 min or I h) with iso-OMPA (100 pM) selectively
inhibited BChE, and these results from human airways were
similar to those previously described (Traina & Serpietri, 1984;
Thomsen et al., 1991).
The data derived from the physiological studies (contractions) as well as results obtained in the biochemical protocols
suggest that the BChE activity detected in human bronchi is
associated with the enzymatic degradation of acetylcholine in
these tissues.
This work was supported in part by l'Association Francaise contre les
Myopathies. The authors thank A. Perrin for technical assistance.

HUMAN AIRWAYS AND BUTYRYLCHOLINESTERASE

919

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(Received April 6, 1992

Revised November 12, 1992
Accepted November 18, 1992)